In U.S. Pat. No. 5,047,321, Loken and Terstappen described the multiparameter analysis of cellular components in a body fluid. The body fluids described included blood and bone marrow. Using a combination of two nucleic acid dyes, a fluorescently labeled monoclonal antibody and two light scatter parameters, Loken and Terstappen were able to discriminate between and among various components of blood and bone marrow, count the number of cells within each component and provide a differential analysis of each. Using a combination of LDS-751 (Exciton) as a DNA dye, Thiazole Orange (‘TO’, Molecular Probes, Inc.) as an RNA dye, a fluorescently labelled anti-CD45 monoclonal antibody and forward and orthogonal light scatter on whole blood or bone marrow aspirates, Loken and Terstappen were able to detect and differentiate between erythrocytes, reticulocytes, nucleated erythrocytes, platelets, lymphocytes, monocytes, neutrophilic granulocytes, basophilic granulocytes, eosinophilic granulocytes and precursors of all nucleated cells but not other normal (i.e dendritic cells) or pathologic (leukemic blasts) leukocytes.
In U.S. Pat. No. 6,287,791, Terstappen and Chen described a further refinement of U.S. Pat. No. 5,047,321, but were not showing any better characterization of the different leukocyte subpopulations.
In U.S. Pat. No. 5,137,809, Loken and Sha described the multiparameter analysis of cellular components in bone marrow. The authors used a combination of monoclonal antibodies labeled with different fluorochromes to stain all leukocytes in a first step and then stain selected subpopulations in a second step.
All the methods referenced above were able to identify the majority of the leukocytes and were only identifying selected subpopulations as identified by the monoclonal antibodies used. Also, given the enumeration of the leukocyte subpopulations in terms of percentage of total leukocytes, it is impossible to link the obtained profile to the results as obtained by the traditional hematology cell counters. Under that condition, the different pathways identified remain isolated from the key diagnostic test that is in common use in all clinical laboratories.
In U.S. Pat. No. 5,627,037, Ward et al. describe a one-step method for the detection and the enumeration of absolute counts of one or more cell populations in a blood sample. The method employs a reagent comprising a mixture of one or more cell markers, a fluorescent microparticle and a fixative. The method refers to the absolute counting of leukocytes, such as CD4+ lymphocytes but does not give any indication of how to enumerate individual leukocyte subpopulations within this sample/reagent mixture.